174 L. M. BLACK 



involves less work than the following one and, when it is available, it would 

 seem to be preferable. 



(2) Transovarial passage of virus from generation to generation until the 

 dilution attained exceeds with certainty the maximum possible without 

 multiphcation in the insect. Black's estimate (1950) that a dilution of IQ-^^ 

 should be exceeded still seems to be of the correct order of magnitude. 



In addition to these two unequivocal methods there are others that can 

 provide evidence indicating with high probability that multiplication occurs. 

 These are: 



(1) Methods of titrating for virus increase in the insect. Demonstrated 

 increases are almost certainly due to multiplication, but this evidence is 

 subject to the possibility that a given quantity of virus may, for some 

 reason, be detectable at a higher dilution at one time than another (Black, 1941 ). 



(2) Cross-protection between strains of virus m the vector. This phenome- 

 non would seem to depend almost certainly upon multiplication in the 

 vector. However, interference between two strains of a plant virus at time of 

 entrance into the host was demonstrated by the work of Beale (1947) and 

 would seem to be a remote alternative possibility to interference through 

 multiplication. 



(3) A cytopathogenic effect in the vector. Symptoms of disease in the cells 

 of the insect almost certainly indicate that virus is multiplying in those cells. 

 However, a toxic effect without multiplication would seem to be a remote 

 possibility. The same considerations also apply to other symptoms of disease. 



(4) Persistence of virus in the vector and frequency of transmission by 

 single insects (Kunkel, 1954), when independent of or not proportional to the 

 length of the acquisition feeding period, have been associated with multi- 

 plication in the vector and stand in marked contrast to the contrary condition 

 in curly top, where the evidence is against multiphcation. 



In addition, there are a number of other kinds of evidence for which 

 multiplication in the vector is the most probable basis, but which are also 

 subject to other interpretations. 



There is abundant evidence to support Kunkel's interpretation of his 

 heat-induced incubation periods in the vector of aster yellows virus, but the 

 phenomenon by itself, without evidence from other kinds of experiments has 

 been variously interpreted. 



.Black (1954, p. 84) has emphasized the long incubation periods associated 

 with all those viruses that have been demonstrated to multiply in the vector. 

 However, Kunkel (1948), on the one hand, has correlated both long and short 

 incubation periods in the vector with long and short incubation periods in the 

 plant where multiplication is known to occur and, on the other hand, Smith 

 and Lea (1946) have given hjrpothetical explanations for both long and short 

 incubation periods, without assuming multiplication in the vector. 



